The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src 527F can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.

The actin ®lament-associated protein of 110 kDa (AFAP-110) is a Src binding partner that represents a potential modulator of actin ®lament integrity in response to cellular signals. Previous reports have demonstrated that AFAP-110 is capable of directly binding and altering actin ®laments. Deletion of the leucine zipper motif of AFAP-110 (AFAP-110 Dlzip ) has been shown to induce a phenotype which resembles Srctransformed cells, by repositioning actin ®laments into rosettes. This deletion also mimics a conformational change in AFAP-110 that is detected in Src-transformed cells. The results presented here indicate that unlike AFAP-110, AFAP-110 Dlzip is capable of activating cellular tyrosine kinases, including Src family members, and that AFAP-110 Dlzip itself is hyperphosphorylated. The newly tyrosine phosphorylated proteins and activated Src-family members appear to be associated with actinrich lamellipodia. A point mutation that alters the SH3-binding motif of AFAP-110 Dlzip prevents it from activating tyrosine kinases and altering actin ®lament integrity. In addition, a deletion within a pleckstrin homology (PH) domain of AFAP-110 Dlzip will also revert its e ects upon actin ®laments. Lastly, dominant-positive RhoA V14 will block the ability of AFAP-110 Dlzip from inducing actin ®lament rosettes, but does not inhibit Src activation. Thus, conformational changes in AFAP-110 enable it to activate cellular kinases in a mechanism requiring SH3 and/or PH domain interactions. We hypothesize that cellular signals which alter AFAP-110 conformation, enable it to activate cellular kinases such as cSrc, which then direct changes in actin ®lament integrity in a Rho-dependent fashion. Oncogene (2001) 20, 6607 ± 6616.

Background: We investigated the roles of p120 catenin, Cdc42, Rac1, and RhoA GTPases in regulating migration of presomitic mesoderm cells in zebrafish embryos. p120 catenin has dual roles: It binds the intracellular and juxtamembrane region of cadherins to stabilize cadherin-mediated adhesion with the aid of RhoA GTPase, and it activates Cdc42 GTPase and Rac1 GTPase in the cytosol to initiate cell motility. Results: During gastrulation of zebrafish embryos, knockdown of the synthesis of zygotic p120 catenind1 mRNAs with a splice-site morpholino caused lateral widening and anterior-posterior shortening of the presomitic mesoderm and somites and a shortened anterior-posterior axis. These phenotypes indicate a cell-migration effect. Co-injection of low amounts of wild-type Cdc42 or wild-type Rac1 or dominant-negative RhoA mRNAs, but not constitutively-active Cdc42 mRNA, rescued these p120 catenin d1-depleted embryos. Conclusions: These downstream small GTPases require appropriate spatiotemporal stimulation or cycling of GTP to guide mesodermal cell migration. A delicate balance of Rho GTPases and p120 catenin underlies normal development. Developmental Dynamics 241:1545-1561, 2012. V C 2012 Wiley Periodicals Inc.Key words: p120 Catenin (CTNND1); ARVCF; Delta-catenin (CTNND2b); Cdc42 GTPase; Rac1 GTPase, RhoA GTPase; gastrulation; presomitic mesoderm; somites; zebrafish Key findings p120 catetin is required for extension of the dorsal axis and normal migration of the presomitic mesoderm. Cdc42 and Rac1 GTPases are downstream of p120 catenin d1 signaling and require exchange of GTP for GDP. Local stimulation of the exchange of GTP for GDP in Cdc42 and Rac GTPases mediates directional migration of the presomitic mesoderm. A balance of the amount of p120 catenin d1 and localized activation or turnover of Cdc42, Rac1, and Rho GTPase are required for normal zebrafish cell migration. Accepted 31 July 2012 Developmental DynamicsABBREVIATIONS Ab antibody ARVCF armadillo repeat gene deleted in velo-cardio-facial syndrome CA constitutively active Chr chromosome C(t) relative amount of RT-PCR product hpf hours post-fertilization DN dominant negative d1 splice-MO antisense morpholino oligonucleotide to the 12 th splice site of zebrafish p120 catenin d1 p120 catenin d1 (CTNND1) also called p120 catenin, Xenopus p120 catenin is a CTNND1 p120 catenin d2b (CTNND2b) also called Delta-catenin Rok1 Rho kinase1 RT reverse transcriptase minus-RT controls without reverse transcriptase qRT-PCR quantitative real-time PCR WT wild-type Xp120 catenin mRNA Xenopus p120 catenin d1 mRNA.Additional Supporting Information may be found in the online version of this article.

AFAP-110 has an intrinsic ability to alter actin filament integrity as an actin filament crosslinking protein. This capability is regulated by a carboxy terminal leucine zipper (Lzip) motif. The Lzip motif facilitates self-association stabilizing the AFAP-110 multimers. Deletion of the Lzip motif (AFAP-110(Deltalzip)) reduces the stability of the AFAP-110 multimer and concomitantly increases its ability to crosslink actin filaments, in vitro, and to activate cSrc and alter actin filament integrity, in vivo. We sought to determine how the Lzip motif regulates AFAP-110 function. Substitution of the c-Fos Lzip motif in place of the AFAP-110 Lzip motif (AFAP-110(fos)) was predicted to preserve the alpha-helical structure while changing the sequence. To alter the structure of the alpha-helix, a leucine to proline mutation was generated in the AFAP-110 alpha-helical Lzip motif (AFAP-110(581P)), which largely preserved the sequence. The helix mutants, AFAP-110(Deltalzip), AFAP-110(fos), and AFAP-110(581P), demonstrated reduced multimer stability with an increased capacity to crosslink actin filaments, in vitro, relative to AFAP-110. An analysis of opposing binding sites indicated that the carboxy terminus/Lzip motif can contact sequences within the amino terminal pleckstrin homology (PH1) domain indicating an auto-inhibitory mechanism for regulating multimer stability and actin filament crosslinking. In vivo, only AFAP-110(Deltalzip) and AFAP-110(581P) were to activate cSrc and to alter cellular actin filament integrity. These data indicate that the intrinsic ability of AFAP-110 to crosslink actin filaments is dependent upon both the sequence and structure of the Lzip motif, while the ability of the Lzip motif to regulate AFAP-110-directed activation of cSrc and changes in actin filament integrity in vivo is dependent upon the structure or presence of the Lzip motif. We hypothesize that the intrinsic ability of AFAP-110 to crosslink actin filaments or activate cSrc are distinct functions.

ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is targeted to the nucleus of infected cells where it binds to chromatin and mediates viral episome persistence, interacts with cellular proteins and plays a role in latency and tumorigenesis. A structurally related LANA homolog has been identified in the retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of KSHV. Here, we report the evolutionary and functional conservation of a novel bi-functional nuclear localization signal (NLS) in KSHV and RFHV LANA. N-terminal peptides from both proteins were fused to EGFP or double EGFP fusions to examine their ability to induce nuclear transport of a heterologous protein. In addition, GST-pull down experiments were used to analyze the ability of LANA peptides to interact with members of the karyopherin family of nuclear transport receptors. Our studies revealed that both LANA proteins contain an N-terminal arginine/glycine (RG)-rich domain spanning a conserved chromatin-binding motif, which binds directly to importin β1 in a RanGTP-sensitive manner and serves as an NLS in the importin β1-mediated non-classical nuclear import pathway. Embedded within this domain is a conserved lysine/arginine-(KR)-rich bipartite motif that binds directly to multiple members of the importin α family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin α/β-mediated nuclear import pathway. The positioning of a classical bipartite kr-NLS embedded within a non-classical rg-NLS is a unique arrangement in these viral proteins, whose nuclear localization is critical to their functionality and to the virus life cycle. The ability to interact with multiple import receptors provides alternate pathways for nuclear localization of LANA. Since different import receptors can import cargo to distinct subnuclear compartments, a multifunctional NLS may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency.